Sensitization of Chemotherapeutic Agent Resistant Neoplastic Cells With a Virus

ABSTRACT

The present invention relates to a method of increasing the sensitivity of neoplastic cells to chemotherapeutic agents by using a virus, a method of treating proliferative disorders with a virus and chemotherapeutic agents, and a method for preventing a neoplasm from developing drug resistance to chemotherapeutic agents. The virus is preferably a reovirus.

RELATED APPLICATION

This application claims the benefit of U.S. Provisional Application Ser.No. 60/270,363, filed Feb. 20, 2001, which is hereby incorporated byreference in its entirety.

FIELD OF THE INVENTION

The present invention relates to a method of increasing the sensitivityof neoplastic cells to chemotherapeutic agents by using a virus, and amethod of treating proliferative disorders with a virus andchemotherapeutic agents. In particular, the virus is a reovirus.

REFERENCES

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All of the above publications, patents and patent applications areherein incorporated by reference in their entirety to the same extent asif the disclosure of each individual publication, patent application orpatent was specifically and individually indicated to be incorporated byreference in its entirety.

BACKGROUND OF THE INVENTION

Cancer is one of the leading causes of death. Although it has been thefocus of medical research for a long period of time, the main cancertherapies to date remain to be surgery, radiation therapy andchemotherapy. Each one of these therapies is subject to limitationswhich are not currently overcome, and the search for an improved therapycontinues.

One significant problem of chemotherapy is that tumors can developresistance to drugs. For example, a drug may be highly effective when itis first introduced to the patient, killing tumor cells and reducing thesize of the tumor such that the patient goes into a remission. However,the tumor may regrow after a period of time, and this time the same drugis not effective at all at killing the regrown tumor cells. Thisphenomenon of progressive drug resistance is believed to be due to asmall population of drug resistant cells in the tumor which survives theinitial drug treatment while the majority of the tumor is killed. Theseresistant cells eventually grow back to form a tumor comprisingessentially only drug resistant cells.

Treatment at the outset with a combination of drugs was proposed as asolution, given the small probability that mutations which lead to twoor more different drug resistance pathways would arise spontaneously inthe same cell (DeVita, Jr., 1983). However, it has been discovered thatcells which are resistant to one drug are often resistant to multipledrugs, including structurally unrelated drugs which are capable ofkilling tumor cells by different pathways. Therefore, combination drugtherapy does not solve the problem.

Although the mechanisms are not completely clear, the best documentedand clinically relevant mechanism for multidrug resistance in tumorcells is correlated with the expression of P-glycoprotein, the productof the MDR1 gene. P-glycoprotein is a broad specificity efflux pumplocated in the cell membrane, and functions by decreasing theintracellular accumulation of many lipophilic cytotoxic drugs, includingsome widely used anticancer agents such as anthracyclines, vincaalkaloids, epipodophyllotoxins, actinomycin D and taxol, therebyrendering cells resistant to these drugs (Pastan et al., 1991).

In addition to MDR1, another pleiotropic drug transporter has then beendiscovered (Grant et al., 1994). This protein, termed the MultidrugResistance-associated Protein (MRP), has been shown to confer a patternof resistance to cytotoxic drugs, particularly chemotherapeutic drugs,similar to the P-glycoprotein transporter encoded by the MDR1 gene.Subsequently, an increasing number of MRP related proteins have beendiscovered (Borst et al., 2000). Each one may have a different drugspecificity, but the physiologic functions are not completely known.

Therefore, the causes of drug resistance are not fully understood andthere is still a need for methods to overcome drug resistance in orderto treat tumors more effectively.

SUMMARY OF THE INVENTION

The present invention provides a method of sensitizing drug resistantcells to chemotherapeutic agents by the use of a virus, particularly areovirus. Reovirus has recently been discovered as a selectiveanticancer agent which kills ras-activated neoplastic cells but notnormal cells, due to selective replication of reovirus in cells with anactivated ras pathway (U.S. Pat. No. 6,136,307; Coffey et al., 1998;Strong et al., 1998). Unexpectedly, it was further discovered in thepresent invention that reovirus increased the sensitivity of cells tochemotherapeutic agents as well. Thus, a tumor which is refractory tocisplatin was treated with a combination of cisplatin and reovirus, andthe results indicate that the combination was more effective thanreovirus alone. Since cisplatin had no effect on the tumor whenadministered in the absence of reovirus, the effect of the combinationwas not simply an additive or synergistic result of the individualeffects. Instead, reovirus sensitized the tumor to a chemotherapeuticagent to which the tumor is normally refractory.

Accordingly, one aspect of the present invention provides a method ofsensitizing a neoplastic cell, comprising

-   -   (a) administering to said cell an effective amount of reovirus;        and    -   (b) administering an effective amount of the chemotherapeutic        agent to said cell.

The cell is preferably a ras-activated neoplastic cell. Most preferably,the reovirus is administered under conditions which result in infectionof the neoplastic cell by the reovirus. The cell may be susceptible tothe chemotherapeutic agent in the absence of reovirus, but it ispreferably refractory to the chemotherapeutic agent.

To sensitize the cell, reovirus can preferably be administered prior toadministration of the chemotherapeutic agent. Alternatively, in anotherpreferred embodiment, reovirus and the chemotherapeutic agent can beadministered concurrently with each other. Both the reovirus andchemotherapeutic agent may individually be administered in a single doseor multiple doses.

The neoplastic cell is preferably located in a mammal, particularly adog, cat, sheep, goat, cattle, horse, pig, human or non-human primates.The cell is most preferably located in a human.

The present invention may be used to sensitize cells to anychemotherapeutic agent. Preferred chemotherapeutic agents include5-fluorouracil, mitomycin C, methotrexate, hydroxyurea,cyclophosphamide, dacarbazine, mitoxantrone, anthracycline (e.g.epirubicin and doxorubicin), carboplatin, cisplatin, taxol, taxotere,tamoxifen, anti-estrogens, and interferons. More preferably, thechemotherapeutic agent is a platinate or taxol. The most preferredchemotherapeutic agent is cisplatin.

The reovirus may be any reovirus, including mammalian and avianreovirus. Preferably, the reovirus is a mammalian virus, particularly ahuman reovirus. The human reovirus is preferably a serotype 3 reovirusand most preferably a Dearing strain serotype 3 reovirus.

Also provided by the present invention is a method of treating a subjectwith a proliferative disorder, said subject comprising neoplastic cellswhich are refractory to a chemotherapeutic agent, comprising:

-   -   (a) administering to the subject an effective amount of reovirus        under conditions which result in infection of the neoplastic        cells by the reovirus; and    -   (b) administering an effective amount of the chemotherapeutic        agent to said subject.

The reovirus may be administered any time with respect to thechemotherapeutic agent. Preferably, the reovirus is administered priorto or concurrently with administration of the chemotherapeutic agent.Preferably the reovirus is administered in multiple doses. Thechemotherapeutic agent may also be administered in multiple doses. It iscontemplated that the reovirus may be administered in multiple dosesprior to any administration of the chemotherapeutic agent.

The subject is preferably a mammal, particularly a dog, cat, sheep,goat, cattle, horse, pig, human or non-human primates, and mostpreferably a human.

The proliferative disorder may be solid tumor, particularly lung cancer,prostate cancer, colorectal cancer, thyroid cancer, renal cancer,adrenal cancer, liver cancer, pancreatic cancer, breast cancer andcentral and peripheral nervous system cancer. To treat the solid tumor,reovirus may be administered, for example, by injection into or near thesolid tumor or by systematic administration.

The proliferative disorder may alternatively be a hematopoietic tumor,particularly lymphomas and leukemias.

The proliferative disorder may be an original tumor or a metastatictumor.

Also provided is a method for preventing a neoplasm in a subject fromdeveloping drug resistance to a chemotherapeutic agent, comprising:

-   -   (a) administering to the subject an effective amount of reovirus        under conditions which result in infection of the neoplasm by        the reovirus; and    -   (b) administering to the subject an effective amount of a        chemotherapeutic agent.

The reovirus may be administered any time with respect to thechemotherapeutic agent. Preferably, the reovirus is administered priorto or concurrently with administration of the chemotherapeutic agent.Preferably the reovirus is administered in multiple doses. Thechemotherapeutic agent may also be administered in multiple doses. It iscontemplated that the reovirus may be administered in multiple dosesprior to any administration of the chemotherapeutic agent.

The subject is preferably a mammal, particularly a dog, cat, sheep,goat, cattle, horse, pig, human or non-human primates, and mostpreferably a human.

Preferably, administration of the reovirus prevents the neoplasm fromdeveloping drug resistance to multiple drugs, including structurallyunrelated drugs. Accordingly, a preferred embodiment of the presentinvention provides a method for preventing a neoplasm in a subject fromdeveloping drug resistance to a chemotherapeutic agent wherein drugresistance to a second chemotherapeutic agent is also prevented, whichmethod comprising:

-   -   (a) administering to the subject an effective amount of reovirus        under conditions which result in infection of the neoplasm by        the reovirus; and    -   (b) administering to the subject an effective amount of a        chemotherapeutic agent.

Other aspects of the present invention provide method of sensitizingneoplastic cells, treating proliferative disorder, or preventingdevelopment of drug resistance using other viruses in the same manner asreovirus. The virus useful in the present invention is preferablycapable of selectively infecting neoplastic cells. Preferably, the virusis selected from the group consisting of modified adenovirus, modifiedHSV, modified vaccinia virus, modified parapoxvirus orf virus, delNS1virus, p53-expressing viruses, the ONYX-015 virus, the Delta24 virus,vesicular stomatitis virus, the herpes simplex virus 1 mutant which isdefective in hrR3, Newcastle disease virus, encephalitis virus, herpeszoster virus, hepatitis virus, influenza virus, varicella virus, andmeasles virus. More preferably, the virus is selected from the groupconsisting of modified adenovirus, modified HSV, modified vacciniavirus, modified parapoxvirus orf virus, delNS1 virus, p53-expressingviruses, the ONYX-015 virus, the Delta24 virus, and vesicular stomatitisvirus.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the effects of reovirus and cisplatin on tumor growth.Animals bearing syngeneic tumors were given mock treatment (Series 1),cisplatin alone (Series 2), reovirus alone (Series 3) or the combinationof cisplatin and reovirus (Series 4). The results indicate that thetumors were refractory to cisplatin. However, in the presence ofreovirus, the tumors became sensitive to cisplatin.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to a method of sensitizing drugresistant cells to chemotherapeutic agents by the use of a virus,particularly a reovirus. Reovirus has recently been discovered as aselective anticancer agent which kills ras-activated neoplastic cellsbut not normal cells, due to selective replication of reovirus in cellswith an activated ras pathway (U.S. Pat. No. 6,136,307; Coffey et al.,1998; Strong et al., 1998). Unexpectedly, it was further discovered inthe present invention that reovirus increased the sensitivity of cellsto chemotherapeutic agents as well. The present invention thus providesa method of enhancing both the efficacy and selectivity of cancerchemotherapy. It may also be used to prevent the development ofprogressive drug resistance.

Prior to describing the invention in further detail, the terms used inthis application are defined as follows unless otherwise indicated.

DEFINITIONS

“Sensitizing” a neoplastic cell to a chemotherapeutic agent, as usedherein, refers to the act of enhancing the sensitivity of a neoplasticcell to a chemotherapeutic agent.

“Sensitivity” of a neoplastic cell to a chemotherapeutic agent is thesusceptibility of the neoplastic cell to the inhibitory effect of thechemotherapeutic agent. For example, sensitivity of a neoplastic cell toa chemotherapeutic agent is indicated by reduction in growth rate of thecell in response to the chemotherapeutic agent. The sensitivity may alsobe demonstrated by a reduction of the symptoms caused by the neoplasticcells.

A neoplastic cell that is “refractory” to a chemotherapeutic agent is aneoplastic cell not killed or growth inhibited by the chemotherapeuticagent. To determine if a neoplastic cell is growth inhibited, the growthrate of the cell in the presence or absence of the chemotherapeuticagent can be determinedly established methods in the art. The neoplasticcell is not growth inhibited by the chemotherapeutic agent if the growthrate is not significantly different with or without the chemotherapeuticagent.

A tumor that is “refractory” to a chemotherapeutic agent is a tumor ofwhich the rate of size increase or weight increase does not change inthe presence of the chemotherapeutic agent. Alternatively, if thesubject bearing the tumor displays similar symptoms or indicators of thetumor whether the subject receives the chemotherapeutic agent or not,the tumor is refractory to the chemotherapeutic agent. For example,white cell count is commonly used as an indicator of leukemia. If thewhite cell count of a leukemia patient does not significantly changeafter receiving a chemotherapeutic drug, the leukemia of this patient isrefractory to the chemotherapeutic drug.

A “neoplastic cell”, also known as a “cell with a proliferativedisorder”, refers to a cell which proliferates at an abnormally highrate. A new growth comprising neoplastic cells is a neoplasm, also knownas a tumor. A neoplasm is an abnormal tissue growth, generally forming adistinct mass, that grows by cellular proliferation more rapidly thannormal tissue growth. A neoplasm may show partial or total lack ofstructural organization and functional coordination with normal tissue.As used herein, a neoplasm is intended to encompass hematopoietic tumorsas well as solid tumors.

A neoplasm may be benign (benign tumor) or malignant (malignant tumor orcancer). Malignant tumors can be broadly classified into three majortypes. Malignant neoplasms arising from epithelial structures are calledcarcinomas, malignant neoplasms that originate from connective tissuessuch as muscle, cartilage, fat or bone are called sarcomas and malignanttumors affecting hematopoietic structures (structures pertaining to theformation of blood cells) including components of the immune system, arecalled leukemias and lymphomas. Other neoplasms include, but are notlimited to neurofibromatosis.

A “proliferative disorder” is a disease or condition caused by cellswhich grow more quickly than normal cells, i.e., neoplastic cells.Proliferative disorders include benign tumors and malignant tumors. Whenclassified by structure of the tumor, proliferative disorders includesolid tumors and hematopoietic tumors.

“Ras-activated neoplastic cells” or “ras-mediated neoplastic cells”refer to cells which proliferate at an abnormally high rate due to, atleast in part, activation of the ras pathway. The ras pathway may beactivated by way of ras gene structural mutation, elevated level of rasgene expression, elevated stability of the ras gene message, or anymutation or other mechanism which leads to the activation of ras or afactor or factors downstream or upstream from ras in the ras pathway,thereby increasing the ras pathway activity. For example, activation ofEGF receptor, PDGF receptor or sos results in activation of the raspathway. Ras-mediated neoplastic cells include, but are not limited to,ras-mediated cancer cells, which are cells proliferating in a malignantmanner due to activation of the ras pathway.

“Infection by reovirus” refers to the entry and replication of reovirusin a cell. Similarly, “infection of a neoplasm by reovirus” refers tothe entry and replication of reovirus in the cells of a neoplasm.

A “chemotherapeutic agent” or “chemotherapeutic drug” is any chemicalcompound used in the treatment of a proliferative disorder. Examples ofchemotherapeutic agents include, without being limited to, the followingclasses of agents:

-   -   nitrogen mustards, e.g. cyclophosphamide, trofosfamide,        ifosfamide and chlorambucil;    -   nitroso ureas, e.g. carmustine (BCNU), lomustine (CCNU),        semustine (methyl. CCNU) and nimustine (ACNU);    -   ethylene imines and methyl-melamines, e.g. thiotepa;    -   folic acid analogs, e.g. methotrexate;    -   pyrimidine analogs, e.g. 5-fluorouracil and cytarabine;    -   purine analogs, e.g. mercaptopurine and azathioprine;    -   vinca alkaloids, e.g. vinblastine, vincristine and vindesine;    -   epipodophyllotoxins, e.g. etoposide and teniposide;    -   antibiotics, e.g. dactinomycin, daunorubicin, doxorubicin,        epirubicin, bleomycin a2, mitomycin c and mitoxantrone;    -   estrogens, e.g. eiethyl stilbestrol;    -   gonadotropin-releasing hormone analogs, e.g. leuprolide,        buserelin and goserelin;    -   antiestrogens, e.g. tamoxifen and aminoglutethimide;    -   androgens, e.g. testolactone and drostanolonproprionate;    -   platinates, e.g. cisplatin and carboplatin; and    -   interferons, including interferon-alpha, beta and gamma.

The chemotherapeutic agents of the present invention are preferablysmall chemical compounds. Thus, the chemotherapeutic agent has amolecular weight of preferably less than about 5,000, more preferablyless than about 3,000, still more preferably less than about 2,000, andmost preferably less than about 1,000.

A “platinate” is a chemotherapeutic drug that contains platinum as acentral atom. Examples of platinates include cisplatin, carboplatin,oxaliplatin, ormaplatin, iproplatin, enloplatin, nedaplatin, ZD0473(cis-amminedichloro(2-methylpyridine)-platinum (II)), BBR3464 and thelike.

“Reovirus” refers to any virus classified in the reovirus genus, whethernaturally occurring, modified or recombinant. Reoviruses are viruseswith a double-stranded, segmented RNA genome. The virions measure 60-80nm in diameter and possess two concentric capsid shells, each of whichis icosahedral. The genome consists of double-stranded RNA in 10-12discrete segments with a total genome size of 16-27 kbp. The individualRNA segments vary in size. Three distinct but related types of reovirushave been recovered from many species. All three types share a commoncomplement-fixing antigen.

The human reovirus consists of three serotypes: type 1 (strain Lang orTIL), type 2 (strain Jones, T2J) and type 3 (strain Dearing or strainAbney, T3D). The three serotypes are easily identifiable on the basis ofneutralization and hemagglutinin-inhibition assays (see, for example,Fields, B. N. et al., 1996).

The reovirus may be naturally occurring or modified. The reovirus is“naturally-occurring” when it can be isolated from a source in natureand has not been intentionally modified by humans in the laboratory. Forexample, the reovirus can be from a “field source”, that is, from ahuman who has been infected with the reovirus.

The reovirus may be modified but still capable of lytically infecting amammalian cell having an active ras pathway. The reovirus may bechemically or biochemically pretreated (e.g., by treatment with aprotease, such as chymotrypsin or trypsin) prior to administration tothe proliferating cells. Pretreatment with a protease removes the outercoat or capsid of the virus and may increase the infectivity of thevirus. The reovirus may be coated in a liposome or micelle (Chandron andNibert, 1998). For example, the virion may be treated with chymotrypsinin the presence of micelle forming concentrations of alkyl sulfatedetergents to generate a new infectious subvirion particle.

The reovirus may be a recombinant (i.e. reassorted) reovirus from two ormore types of reoviruses with differing pathogenic phenotypes such thatit contains different antigenic determinants, thereby reducing orpreventing an immune response by a mammal previously exposed to areovirus subtype. Such recombinant virions can be generated byco-infection of mammalian cells with different subtypes of reovirus withthe resulting resorting and incorporation of different subtype coatproteins into the resulting virion capsids.

The reovirus may be modified by incorporation of mutated coat proteins,such as for example σ1, into the virion outer capsid. The proteins maybe mutated by replacement, insertion or deletion. Replacement includesthe insertion of different amino acids in place of the native aminoacids. Insertions include the insertion of additional amino acidresidues into the protein at one or more locations. Deletions includedeletions of one or more amino acid residues in the protein. Suchmutations may be generated by methods known in the art. For example,oligonucleotide site directed mutagenesis of the gene encoding for oneof the coat proteins could result in the generation of the desiredmutant coat protein. Expression of the mutated protein in reovirusinfected mammalian cells in vitro such as COS1 cells will result in theincorporation of the mutated protein into the reovirus virion particle(Turner and Duncan, 1992; Duncan et al., 1991; Mah et al., 1990).

The reovirus is preferably a reovirus modified to reduce or eliminate animmune reaction to the reovirus. Such modified reovirus are termed“immunoprotected reovirus”. Such modifications could include packagingof the reovirus in a liposome, a micelle or other vehicle to mask thereovirus from the mammals immune system. Alternatively, the outer capsidof the reovirus virion particle may be removed since the proteinspresent in the outer capsid are the major determinant of the hosthumoral and cellular responses.

The term “attenuated adenovirus” or “modified adenovirus” means anadenovirus in which the gene product or products which prevents theactivation of PKR is lacking, inhibited or mutated such that PKRactivation is not blocked. Preferably, the VAI RNA's are nottranscribed. Such attenuated or modified adenovirus would not be able toreplicate in normal cells that do not have an activated ras pathway,however, it would be able to infect and replicate in cells having anactivated ras pathway.

The term “attenuated HSV” or “modified HSV” means a herpes simplex virus(HSV) in which the gene product or products that prevents the activationof PKR is lacking, inhibited or mutated such that PKR activation is notblocked. Preferably, the HSV gene _(γ1)34.5 is not transcribed. Suchattenuated or modified HSV would not be able to replicate in normalcells that do not have an activated ras pathway, however, it would beable to infect and replicate in cells having an activated ras pathway.

“Parapoxvirus orf virus” is a poxvirus. It is a virus that induces acutecutaneous lesions in different mammalian species, including humans.Parapoxvirus orf virus naturally infects sheep, goats and humans throughbroken or damaged skin, replicates in regenerating epidermal cells andinduces pustular leasions that turn to scabs (Haig et al., 1998). Theterm “attenuated parapoxvirus off virus” or “modified parapoxvirus orfvirus” means a parapoxvirus orf virus in which the gene product orproducts which prevents the activation of PKR is lacking, inhibited ormutated such that PKR activation is not blocked. Preferably, the geneOV20.0L is not transcribed. Such attenuated or modified parapoxvirus orfvirus would not be able to replicate in normal cells that do not have anactivated ras pathway, however, it would be able to infect and replicatein cells having an activated ras pathway.

The term “attenuated vaccinia virus” or “modified vaccinia virus” meansa vaccinia virus in which the gene product or products which preventsthe activation of PKR is lacking, inhibited or mutated such that PKRactivation is not blocked. Preferably, the E3L gene and/or the K3L geneis not transcribed. Such attenuated or modified vaccinia virus would notbe able to replicate in normal cells that do not have an activated raspathway, however, it would be able to infect and replicate in cellshaving an activated ras pathway.

“Administration of reovirus” to a subject refers to the act ofadministering reovirus to a subject in a manner so that it contacts thetarget neoplastic cells. The route by which the reovirus isadministered, as well as the formulation, carrier or vehicle, willdepend on the location as well as the type of the target cells. A widevariety of administration routes can be employed and is discussed belowin further detail.

“Treating a proliferative disorder” means alleviating or eliminating thesymptoms of a proliferative disorder, or slowing down the progress of aproliferative disorder.

A “metastatic tumor” is a tumor that has metastasized from a tumorlocated at another place in the same animal.

An “effective amount” is an amount of a chemotherapeutic agent orreovirus which is sufficient to result in the intended effect. For achemotherapeutic agent used to treat a disease, an efficient amount isan amount sufficient to alleviate or eliminate the symptoms of thedisease, or to slow down the progress of the disease. For a reovirus tosensitize a tumor to a chemotherapeutic agent, an efficient amount is anamount sufficient to increase sensitivity of the tumor to thechemotherapeutic agent.

“Progressive drug resistance” refers to the phenomenon wherein aneoplasm is initially susceptible to a chemotherapeutic agent, but theefficacy of the agent in inhibiting neoplastic growth or reducingsymptoms of the disease decreases over time.

Methods

Reovirus is an effective therapeutic agent against ras-activatedneoplasia because it selectively replicates in cells with an activatedras pathway (U.S. Pat. No. 6,136,307). The ras pathway is not activatedin normal cells, therefore reovirus kills neoplastic cells with highselectivity. Without being limited to a theory, it is thought that viralgene transcription in normal cells correlated with phosphorylation of acellular protein of approximately 65 kDa, determined to bedouble-stranded RNA-activated protein kinase (PKR), that was notobserved in ras-activated cells. Phosphorylation of PKR leads toinhibition of translation, therefore viral replication can not becompleted. In ras-activated cells, however, ras or its downstreamfactors blocks the phosphorylation of PKR, thereby allowing viraltranslation and replication to go on.

In the present invention, we treated tumors with a combination ofreovirus and chemotherapeutic agents. Unexpectedly, we found thatreovirus was capable of sensitizing neoplastic cells to chemotherapeuticagents, whereas the chemotherapeutic agents had no effect on the cellswhen administered alone. As shown in FIG. 1, C3H10T1/2 derived tumorswere refractory to cisplatin. These tumors grew aggressively in thepresence of cisplatin at a growth rate that was essentially the same asuntreated tumors. In contrast, the tumors treated with both reovirus andcisplatin almost completely stopped growing. The inhibitory effect ofthe combination (reovirus plus cisplatin) was much higher than reovirusalone, indicating that cisplatin contributed to the killing of tumorcells. Therefore, while the cells are refractory to cisplatin, reovirustreatment increased the sensitivity of the tumor cells to the drug.

Without being limited to a theory, we believe that reovirus sensitizestumor cells to chemotherapeutic agents by enhancing accumulation of theagents in tumor cells, or by inducing apoptosis. Reovirus is known toinhibit protein synthesis of the host cell in favor of translation ofits own proteins. Therefore, reovirus infection may inhibit thesynthesis of drug transporter proteins, such as MDR1 or the MRPs, andenable drugs to accumulate in the cell. Since drug transporter proteinsare responsible for transporting various drugs out of the cell,including structurally unrelated drugs, inhibiting the synthesis of suchtransporter proteins would lead to sensitization of the cell to avariety of drugs. Alternatively, reovirus is known to induce apoptosisof the infected cells, which may render the cells more susceptible tofurther stress.

The present invention thus provides a valuable method of increasing boththe efficacy and selectivity of chemotherapy. Selectivity has been amajor problem with chemotherapy, because chemotherapeutic agentsgenerally inhibit the growth of both normal cells and neoplastic cells.Chemotherapeutic agents do display limited selectivity, however, sinceneoplastic cells grow faster than most normal cells and hence are growthinhibited to a greater extent. Nevertheless, fast growing normal cells,such as bone marrow cells, tend to be severely damaged bychemotherapeutic drugs, leading to significant side effects. Incontrast, reovirus is highly selective for neoplastic cells andsensitizes neoplastic cells only. Thus, reovirus enhances theaccumulation of chemotherapeutic drugs only in neoplastic cells, therebyincreasing both efficacy and selectivity of the chemotherapeutic agents.

In the present invention, it is preferable that reovirus increasessensitivity of cells or animals to the drug by at least about 20% ascompared to the effect of the drug in the absence of reovirus. Theincrease in sensitivity is more preferably at least about 40%, yet morepreferably at least about 70%, and even more preferably at least about100%. In the most preferred embodiment, as in Example 1, reovirus isuseful to sensitize a cell which is refractory to the drug in theabsence of reovirus, and the sensitization effect cannot be numericallyexpressed.

The sensitivity of a cell to a drug can be observed or measuredaccording to established methods in the art, which may vary with thenature of the disease. For example, sensitivity of a neoplastic cell toa drug may be determined by the size of the tumor or growth rate of theneoplastic cell (for instance see Example 1). Sensitivity may also beobserved as reduction of the cognate symptoms or disease indicators,such as blood cell count in leukemia patients or liver function inhepatoma patients.

The present invention can be used to increase the sensitivity ofneoplastic cells to any chemotherapeutic agents. Preferredchemotherapeutic agents include 5-fluorouracil, mitomycin C,methotrexate, hydroxyurea, cyclophosphamide, dacarbazine, mitoxantrone,anthracyclins (e.g., epirubicin and doxurubicin), carboplatin,cisplatin, taxol, taxotere, tamoxifen, anti-estrogens, and interferons.While new chemotherapeutic agents continue to be developed, it isexpected that drug resistance to the new agents will also occur in thesame manner as resistance to the known agents. Accordingly, reovirus isexpected to sensitize neoplastic cells to the new chemotherapeuticagents, or to prevent neoplasia from developing drug resistance to thenew agents. A skilled artisan will be able to determine if the presentmethod applies to the new agents according to methods disclosed herein.

The reovirus is administered in a manner so that it contacts the targetneoplastic cells. The route by which the reovirus is administered, aswell as the formulation, carrier or vehicle, will depend on the locationas well as the type of the target cells. A wide variety ofadministration routes can be employed. For example, for a solid neoplasmthat is accessible, the reovirus can be administered by injectiondirectly to the neoplasm. For a hematopoietic neoplasm, for example, thereovirus can be administered intravenously or intravascularly. Forneoplasms that are not easily accessible within the body, such asmetastases, the reovirus is administered in a manner such that it can betransported systemically through the body of the mammal and therebyreach the neoplasm (e.g., intravenously or intramuscularly).Alternatively, the reovirus can be administered directly to a singlesolid neoplasm, where it then is carried systemically through the bodyto metastases. The reovirus can also be administered subcutaneously,intraperitoneally, intrathecally (e.g., for brain tumor), topically(e.g., for melanoma), orally (e.g., for oral or esophageal neoplasm),rectally (e.g., for colorectal neoplasm), vaginally (e.g., for cervicalor vaginal neoplasm), nasally or by inhalation spray (e.g., for lungneoplasm).

The reovirus or chemotherapeutic agent can be administered in a singledose, or multiple doses (i.e., more than one dose). The multiple dosescan be administered concurrently at different sites or by differentroutes, or consecutively (e.g., over a period of days or weeks). Thereovirus is preferably administered prior to or concurrently withadministration of the chemotherapeutic agent.

The reovirus is preferably formulated in a unit dosage form, each dosagecontaining from about 10² pfus to about 10¹³ pfus of the reovirus. Theterm “unit dosage forms” refers to physically discrete units suitable asunitary dosages for human subjects and other mammals, each unitcontaining a predetermined quantity of reovirus calculated to producethe desired therapeutic effect, in association with a suitablepharmaceutical excipient.

It is contemplated that the present invention may be combined with othertumor therapies such as radiation therapy or surgery.

In addition, the present invention provides a method for preventing aneoplasm from developing drug resistance. Progressive drug resistance isdeveloped by treating a neoplasm with a drug which kills the drugsensitive cells within the neoplasm, thereby selecting the drugresistant cells. Upon expansion of the drug resistant cells, theneoplasm manifests the phenotype of drug resistance. Accordingly,reovirus can be used to sensitize the neoplasm at the onset of thecourse of chemotherapy such that all cells are killed or inhibited,including the drug resistant cells. Therefore, the neoplasm so treatedwould have no opportunity to develop drug resistance.

A cell which is resistant to one drug is often resistant to another drugdue to the phenomenon of multiple drug resistance. Therefore, reovirusis preferably administered to a neoplasm which has not been treated withany chemotherapeutic agent in order to prevent the development of drugresistance. Once drug resistance has developed, however, reovirus canstill be used to sensitize the drug resistant cells and increase theefficacy and selectivity of chemotherapy, as well as directly killingthe neoplastic cells by oncolysis.

As noted above, we believe that reovirus sensitizes neoplastic cells tochemotherapeutic agents by inhibiting host cell protein synthesis orinducing apoptosis. Therefore, it is contemplated that other viruses canalso be used in the same manner as reovirus. In particular, the virusesthat selectively infect neoplastic cells are preferred. These virusesinclude, but are not limited to, modified adenovirus, modified HSV,modified vaccinia virus, modified parapoxvirus on virus, delNS1 virus,p53-expressing viruses, the ONYX-015 virus, the Delta24 virus, vesicularstomatitis virus, the herpes simplex virus 1 mutant which is defectivein hrR3, Newcastle disease virus, encephalitis virus, herpes zostervirus, hepatitis virus, influenza virus, varicella virus, and measlesvirus. These “oncolytic” viruses are discussed below.

Adenovirus, HSV, vaccinia virus, and parapoxvirus orf virus are viruseswhich have developed a mechanism to overcome the double stranded RNAkinase (PKR). Normally, when virus enters a cell, PKR is activated andblocks protein synthesis, and the virus can not replicate in this cell.However, adenovirus makes a large amount of a small RNA, VA1 RNA. VA1RNA has extensive secondary structures and binds to PKR in competitionwith the double stranded RNA (dsRNA) which normally activates PKR. Sinceit requires a minimum length of dsRNA to activate PKR, VA1 RNA does notactivate PKR. Instead, it sequesters PKR by virtue of its large amount.Consequently, protein synthesis is not blocked and adenovirus canreplicate in the cell. It should be noted, however, that although theprotein synthesis machinery is not blocked, host cell protein synthesisis inhibited by the virus to facilitate viral protein synthesis.

Vaccinia virus encodes two gene products, K3L and E3L, whichdown-regulate PKR with different mechanisms. The K3L gene product haslimited homology with the N-terminal region of eIF-2α, the naturalsubstrate of PKR, and may act as a pseudosubstrate for PKR. The E3L geneproduct is a dsRNA-binding protein and apparently functions bysequestering activator dsRNAs.

Similarly, herpes simplex virus (HSV) gene _(γ1)34.5 encodes the geneproduct infected-cell protein 34.5 (ICP34.5) that can prevent theantiviral effects exerted by PKR. The parapoxvirus orf virus encodes thegene OV20.0L that is involved in blocking PKR activity. Thus, theseviruses can successfully infect cells without being inhibited by PKR.

In the modified adenovirus, modified HSV, modified vaccinia virus, ormodified parapoxvirus orf virus, the viral anti-PKR mechanism has beenmutated or otherwise inactivated. Therefore, these modified viruses arenot capable of replicating in normal cells which have normal PKRfunction. Ras-activated neoplastic cells, however, are not subject toprotein synthesis inhibition by PKR, because ras inactivates PKR. Thesecells are therefore susceptible to infection by the modified adenovirus,modified HSV, modified vaccinia virus, or modified parapoxvirus orfvirus.

The viruses can be modified or mutated according to the knownstructure-function relationship of the viral PKR inhibitors. Forexample, since the amino terminal region of E3 protein of the vacciniavirus interacts with the carboxy-terminal region domain of PKR, deletionor point mutation of this domain prevents anti-PKR function (Chang etal., 1992, 1993; 1995; Sharp et al., 1998; Romano et al., 1998). The K3Lgene of vaccinia virus encodes pK3, a pseudosubstrate of PKR. There is aloss-of-function mutation within K3L. By either truncating or by placingpoint mutations within the C-terminal portion of K3L protein, homologousto residues 79 to 83 in eIF-2α abolish PKR inhibitory activity(Kawagishi-Kobayashi et al., 1997).

The modified HSV include, but are limited to, R3616 (both copies of the_(γ1)34.5 gene have been deleted), R4009 (two stop codons have beeninserted in the _(γ1)34.5 gene), and G207 (mutated in the ribonucleotidereductase and the _(γ1)34.5 genes) (Andreansky et al., 1996). Thesemodified viruses have been used in brain tumor therapy, and it has beenrecently shown that R3616 preferentially infects ras-activated cells(Farassati et al., 2001).

Similarly, the delNS1 virus (Bergmann et al., 2001) is a geneticallyengineered influenza A virus that can selectively replicate inras-activated neoplastic cells. The NS1 protein of influenza virus is avirulence factor that overcomes the PKR-mediated antiviral response bythe host. NS1 is knocked out in the delNS1 virus, which fails to infectnormal cells, presumably due to PKR-mediated inhibition, but replicatessuccessfully in ras-activated neoplastic cells. Therefore, a modifiedinfluenza virus in which NS1 is modified or mutated, such as the delNS1virus, is also useful in the present invention.

Other oncolytic viruses include the viruses which selectively killneoplastic cells by carrying a tumor suppressor gene. For example, p53is a cellular tumor suppressor which inhibits uncontrolled proliferationof normal cells. However, approximate half of all tumors have afunctionally impaired p53 and proliferate in an uncontrolled manner.Therefore, a virus which expresses the wild type p53 gene canselectively kill the neoplastic cells which become neoplastic due toinactivation of the p53 gene product. Such a virus has been constructedand shown to induce apoptosis in cancer cells that express mutant p53(Blagosklonny et al., 1996).

A similar approach involves viral inhibitors of tumor suppressors. Forexample, certain adenovirus, SV40 and human papilloma virus includeproteins that inactivate p53, thereby allowing their own replication(Nemunaitis 1999). For adenovirus serotype 5, this protein is a 55 Kdprotein encoded by the E1B region. If the E1B region encoding this 55 kdprotein is deleted, as in the ONYX-015 virus (Bischoff et al, 1996;Heise et al., 2000; WO 94/18992), the 55 kd p53 inhibitor is no longerpresent. As a result, when ONYX-015 enters a normal cell, p53 functionsto suppress cell proliferation as well as viral replication, whichrelies on the cellular proliferative machinery. Therefore, ONYX-015 doesnot replicate in normal cells. On the other band, in neoplastic cellswith disrupted p53 function, ONYX-015 can replicate and eventually causethe cell to die. Accordingly, this virus can be used to selectivelyinfect and kill p53-deficient neoplastic cells. A person of ordinaryskill in the art can also mutate and disrupt the p53 inhibitor gene inadenovirus 5 or other viruses according to established techniques.

Another example is the Delta24 virus which is a mutant adenoviruscarrying a 24 base pair deletion in the E1A region (Fueyo et al., 2000).This region is responsible for binding to the cellular tumor suppressorRb and inhibiting Rb function, thereby allowing the cellularproliferative machinery, and hence virus replication, to proceed in anuncontrolled fashion. Delta24 has a deletion in the Rb binding regionand does not bind to Rb. Therefore, replication of the mutant virus isinhibited by Rb in a normal cell. However, if Rb is inactivated and thecell becomes neoplastic, Delta24 is no longer inhibited. Instead, themutant virus replicates efficiently and lyses the Rb-deficient cell.

Yet other oncolytic viruses include the interferon sensitive viruses.Vesicular stomatitis virus (VSV) selectively kills neoplastic cells inthe presence of interferon. Interferons are circulating factors whichbind to cell surface receptors which ultimately lead to both anantiviral response and an induction of growth inhibitory and/orapoptotic signals in the target cells. Although interferons cantheoretically be used to inhibit proliferation of tumor cells, thisattempt has not been very successful because of tumor-specific mutationsof members of the interferon pathway.

However, by disrupting the interferon pathway to avoid growth inhibitionexerted by interferon, tumor cells may simultaneously compromise theiranti-viral response. Indeed, it has been shown that VSV, an enveloped,negative-sense RNA virus rapidly replicated in and killed a variety ofhuman tumor cell lines in the presence of interferon, while normal humanprimary cell cultures were apparently protected by interferon. Anintratumoral injection of VSV also reduced tumor burden of nude micebearing subcutaneous human melanoma xenografts (Stojdl et al., 2000).

Other interferon-sensitive viruses (WO 99/18799), namely viruses whichdo not replicate in a normal cell in the presence of interferons, can beidentified by growing a culture of normal cells, contacting the culturewith the virus of interest in the presence of varying concentrations ofinterferons, then determining the percentage of cell killing after aperiod of incubation. Preferably, less than 20% normal cells is killedand more preferably, less than 10% is killed.

It is also possible to take advantage of the fact that some neoplasticcells express high levels of an enzyme and construct a virus which isdependent on this enzyme. For example, ribonucleotide reductase isabundant in liver metastases but scarce in normal liver. Therefore, aherpes simplex virus 1 (HSV-1) mutant which is defective inribonucleotide reductase expression, hrR3, was shown to replicate incolon carcinoma cells but not normal liver cells (Yoon et al., 2000).

In addition to the viruses discussed above, a variety of other viruseshave been associated with tumor killing, although the underlyingmechanism is not always clear. Newcastle disease virus (NDV) replicatespreferentially in malignant cells, and the most commonly used strain is73-T (Reichard et al., 1992; Zorn et al, 1994; Bar-Eli et al, 1996).Clinical antitumor activities wherein NDV reduced tumor burden afterintratumor inoculation were also observed in a variety of tumors,including cervical, colorectal, pancreas, gastric, melanoma and renalcancer (WO 94/25627; Nemunaitis, 1999).

Moreover, encephalitis virus was shown to have an oncolytic effect in amouse sarcoma tumor, but attenuation may be required to reduce itsinfectivity in normal cells. Tumor regression have been described intumor patients infected with herpes zoster, hepatitis virus, influenza,varicella, or measles virus (for a review, see Nemunaitis, 1999).According to the methods disclosed herein, a person of ordinary skill inthe art can test the ability of these or other viruses to sensitizeneoplastic cells to chemotherapeutic agents, or to prevent a neoplasmfrom developing drug resistance.

The following examples are offered to illustrate this invention and arenot to be construed in any way as limiting the scope of the presentinvention.

EXAMPLES

In the examples below, the following abbreviations have the followingmeanings. Abbreviations not defined have their generally acceptedmeanings.

° C.=degree Celsius

hr=hour

min=minute

μM=micromolar

mM=millimolar

M=molar

ml=milliliter

μl=microliter

mg=milligram

μg=microgram

PAGE=polyacrylamide gel electrophoresis

rpm=revolutions per minute

FBS=fetal bovine serum

DTT=dithiothrietol

SDS=sodium dodecyl sulfate

PBS=phosphate buffered saline

DMEM=Dulbecco's modified Eagle's medium

α-MEM=α-modified Eagle's medium

β-ME=β-mercaptoethanol

MOI=multiplicity of infection

PFU or pfu=plaque forming units

PKR=double-stranded RNA activated protein kinase

EGF=epidermal growth factor

PDGF=platelet derived growth factor

DMSO=dimethylsulfoxide

MDR=multiple drug resistance

MRP=multidrug resistance-associated protein

HSV=herpes simplex virus

Example 1 Sensitization of Refractory Tumor Cells to Cisplatin byReovirus

C3H mice (Charles River) were implanted subcutaneously with 1.0×10⁶ PFUsras-transformed C3H cells (a gift of D. Edwards, University of Calgary)and allowed to develop tumors. The Dearing strain of reovirus serotype 3used in these studies was propagated in suspension cultures of L cellsand purified according to Smith (Smith et al., 1969) with the exceptionthat β-mercaptoethanol (β-ME) was omitted from the extraction buffer.The particle/PFU ratio for purified reovirus was typically 100/1.

The tumor bearing mice were treated with 4 different regimes asdescribed below:

Series No. Reovirus Drug 1 control control 2 control cisplatin 3reovirus control 4 reovirus cisplatin

For animals which received reovirus (Series 3-4), 5×10⁸ PFUs of reovirus(in 20 μl of saline) were injected intravenously via the tail vein ofthe animals on Days 0, 6, 12, and 18. The animals which did not receivereovirus (Series 1-2) were injected with 20 μl of saline in the samemanner and time course. Cisplatin was injected into the tail vein onDays 10, 16 and 22 at a dose of 2.5 mg per kilogram of body weight. Thetumors were measured daily from Day 0 to assess growth rate of thetumors.

The tumors were refractory to cisplatin. As shown in FIG. 1, tumorstreated with cisplatin alone (Series 2) progressed almostindistinguishably from the control tumors (Series 1), indicating thatcisplatin had essentially no inhibitory effects on the growth rate ofthe tumors. In contrast, the combination of cisplatin and reovirus(Series 4) significantly reduced tumor growth. The level of inhibitionby the combination was much more profound than reovirus alone (Series3). Therefore, cisplatin contributed to tumor suppression when used inconjunction with reovirus.

1-34. (canceled)
 35. A method of sensitizing a neoplastic cell to achemotherapeutic agent, comprising: (a) administering to said neoplasticcell an effective amount of a virus, said virus being capable ofselectively infecting neoplastic cells; and (b) administering aneffective amount of the chemotherapeutic agent to said cell.
 36. Themethod of claim 35, wherein the virus is selected from the groupconsisting of modified adenovirus, modified HSV, modified vacciniavirus, modified parapoxvirus orfvirus, delNS1 virus, p53-expressingviruses, ONYX-015, Delta24, and vesicular stomatitis virus.
 37. Themethod of claim 35, wherein the virus is administered prior to theadministration of the chemotherapeutic agent.
 38. The method of claim35, wherein the virus is administered concurrently with thechemotherapeutic agent.
 39. The method of claim 35, wherein theneoplastic cell is located in a mammal.
 40. The method of claim 39,wherein the mammal is selected from the group consisting of dogs, cats,sheep, goats, cattle, horses, pigs, humans and non-human primates. 41.The method of claim 35, wherein the chemotherapeutic agent is selectedfrom the group consisting of 5-fluorouracil, mitomycin C, methotrexate,hydroxyurea, cyclophosphamide, dacarbazine, mitoxantrone, anthracyclins,carboplatin, cisplatin, taxol, taxotere, tamoxifen, anti-estrogens, andinterferons.
 42. The method of claim 41, wherein the chemotherapeuticagent is cisplatin.
 43. A method of treating a subject with achemotherapeutic agent, wherein said subject has a proliferativedisorder and neoplastic cells that are refractory to saidchemotherapeutic agent, comprising: (a) administering to the subject aneffective amount of a virus under conditions that result in infection ofthe neoplastic cells by the virus; and (b) administering an effectiveamount of the chemotherapeutic agent to said subject.
 44. The method ofclaim 43, wherein the virus is selected from the group consisting ofmodified adenovirus, modified HSV, modified vaccinia virus, modifiedparapoxvirus orfvirus, delNS1 virus, pS3-expressing viruses, ONYX-OIS,Delta24, and vesicular stomatitis virus.
 45. The method of claim 43,wherein the virus is administered prior to the administration of thechemotherapeutic agent.
 46. The method of claim 43, wherein the virus isadministered concurrently with the chemotherapeutic agent.
 47. Themethod of claim 43, wherein the virus is administered in multiple doses.48. The method of claim 43, wherein the virus is administered inmultiple doses prior to the administration of the chemotherapeuticagent.
 49. The method of claim 43, wherein the subject is a mammal. 50.The method of claim 43, wherein the mammal is selected from the groupconsisting of dogs, cats, sheep, goats, cattle, horses, pigs, humans andnon-human primates.
 51. The method of claim 50, wherein thechemotherapeutic agent is selected from the group consisting of5-fluorouracil, mitomycin C, methotrexate, hydroxyurea,cyclophosphamide, dacarbazine, mitoxantrone, anthracyclins, carboplatin,cisplatin, taxol, taxotere, tamoxifen, anti-estrogens, and interferons.52. The method of claim 51, wherein the chemotherapeutic agent iscisplatin.
 53. The method of claim 43, wherein the proliferativedisorder is a solid tumor.
 54. The method of claim 53, wherein the solidtumor is selected from the group consisting of lung cancer, prostatecancer, colorectal cancer, thyroid cancer, renal cancer, adrenal cancer,liver cancer, pancreatic-cancer, breast cancer and central andperipheral nervous system cancer.
 55. The method of claim 53, whereinthe virus is administered into or near the solid tumor.
 56. The methodof claim 43, wherein the virus is administered systemically.
 57. Themethod of claim 43, wherein the proliferative disorder is ahematopoietic tumor.
 58. The method of claim 57, wherein thehematopoietic tumor is selected from the group consisting of lymphomasand leukemias.
 59. The method of claim 43, wherein the proliferativedisorder is a metastatic tumor.